Abstract: Provided are methods for analyzing properties of a target portion of a polynucleotide using a nanopore. Also provided are methods for nanopore-based analysis of a polynucleotide, the methods comprising modifying a polynucleotide to comprise an enzyme binding site, an enzyme activity blocking structure, and an enzyme displacement region. Such methods further comprise the use of an enzyme that translocates the polynucleotide through a nanopore against a voltage force when the enzyme activity blocking structure is removed. The enzyme blocking structure is reconstituted by movement of the enzyme. Also provided are methods for providing a polynucleotide comprising an abasic region, a region to be analyzed, a GQ fold, and an enzyme binding site, where such methods further comprise removing one enzyme molecule at the abasic region and allowing another enzyme molecule to bind at the enzyme binding site.

Abstract: Provided are polynucleotide constructs useful in a nanopore analysis using enzyme activity. The polynucleotide constructs include a strand portion of interest to be analyzed in the nanopore analysis and having a 5? end and a 3? end, and a folded sequence located 3? or 5? of and adjacent to an end of the strand portion of interest, where the folded sequence can block the activity of a processive enzyme. In some embodiments, the polynucleotide constructs further include an enzyme binding sequence located 3? of and adjacent to an end of the folded sequence, and an enzyme displacement sequence located 5? of the strand portion of interest. Such polynucleotide constructs further include a threading sequence located 5? of the enzyme displacement sequence and at a 5? terminal end of the polynucleotide construct, where the processive enzyme has a processive activity in a direction from 3? to 5? on the polynucleotide construct.

Abstract: Provided are polynucleotide constructs useful in a nanopore analysis using enzyme activity. The polynucleotide constructs include a strand portion of interest to be analyzed in the nanopore analysis and having a 5? end and a 3? end, and a folded sequence located 3? or 5? of and adjacent to an end of the strand portion of interest, where the folded sequence can block the activity of a processive enzyme. In some embodiments, the polynucleotide constructs further include an enzyme binding sequence located 3? of and adjacent to an end of the folded sequence, and an enzyme displacement sequence located 5? of the strand portion of interest. Such polynucleotide constructs further include a threading sequence located 5? of the enzyme displacement sequence and at a 5? terminal end of the polynucleotide construct, where the processive enzyme has a processive activity in a direction from 3? to 5? on the polynucleotide construct.

Abstract: Disclosed are methods and materials for rereading individual single-stranded DNA or RNA molecules with a nanopore device. A single strand polymer can be passed through a nanopore in a nanopore sequencing device in a first direction and then passed through the pore of the device in the reverse direction. The method uses adapters ligated to the DNA strand, at either end of the segment to be analyzed. The method also uses an enzyme that can move the strand through the nanopore against the force applied by a voltage. When the enzyme is removed from the stand, the strand moves in the opposite direction through the nanopore, under force from the voltage. The 3? adapter contains a sequence that folds into a secondary structure that inhibits the enzyme from translocating on the strand, such as an intramolecular G-quadruplex (GQ). The 5? adapter contains a sequence that prevents binding of the enzyme to the strand.